I Helicobacter pylori genes involved in immune-system evasion We developed and validated the use of a H. pylori specific antibiotic-based in vivo expression technology (IVET) system to identify in vivo induced (ivi) genes in mice and cultured macrophage. We constructed unique H. pylori promoter-screening vectors (pIVET11 and pIVET12). From these genetic screens, we found 31 novel in vivo induced genes (ivi genes) and virulence factors cagA and vacA which were previously reported. These novel ivi genes belong to several functional categories: motility/chemotaxis, LPS synthesis, outer membrane proteins, protein synthesis, oxidative stress response, cell division, transposases, DNA modification, acid resistance, hypothetical proteins and pathogenesis. Based on RT-PCR analysis, the expression levels of the identified ivi genes are upregulated 2-20 fold in H. pylori that were phagocytized by RAW 264.7. Currently, we are focused on three novel ivi genes;flhF, priA, and tnpB (IS606). These in vivo induced genes may be essential for colonization or pathogenesis of the stomach mucosa. RT-PCR experiments confirmed the in vivo induction of these ivi genes. Cloning, expression and construction of null H. pylori mutants of these novel ivi genes is complete. In vitro and in vivo studies of these H. pylori mutant strains are ongoing to determine if these new ivi genes play a role in stomach colonization. II Host mucosal and immune responses to Helicobacter pylori infection We are using several mouse models to study H. pylori infection, pathogenic mechanism(s) and mucosal-immune responses. We selected wild type (wt) C57BL/6 and knockout mice to evaluate the role of several endogenous gene products on the regulation of mucosal immune response to H. pylori infection. The knockout mice allowed us to examine the in vivo role of a number of genes (or gene products) on bacteria load per gram of stomach tissue and pathology. We monitored histological changes by pathology scoring. In collaboration with Dr. Richard DiPaolo, Saint Louis University Medical School, we developed a new technique that allows us to isolate the inflammatory cells from the stomach mucosa of mice. Using this technique, we identify, quantify, and determine the functions and specificities of cells infiltrating the stomach after infection. We observed a 7-fold increase of CD4+ and a 6-fold increase of CD8+ T cells infiltrating the stomach tissue of wt infected mice. The functional assays allowed us to examine the CD4+ and CD8+ T cell responses in the stomach after H. pylori infection. For example, using this technique we confirmed a previously reported observation that T cells infiltrating the stomach produce IFN (a T-helper type 1 response). We also made a new observation that there are T cells producing IL-17, and T cells producing both IL-17 and IFN. The discovery of IL-17 producing T cells suggests an additional T-helper type 17 response to H. pylori infection. This is an important finding because IL-17 production has been associated with the recruitment of neutrophils, causing and sustaining tissue damage related to various autoimmune disorders. This finding provides a link between H. pylori infection and autoimmune gastritis. We are also studying innate and adaptive immunity as they relate to H. pylori infection. The cellular and molecular mechanisms that initiate H. pylori adaptive immunity and T-cell response are poorly understood. Dendritic cells are central to the initiation of adaptive immunity. We initiated in vitro studies to compare immune responses to H. pylori infection involving a common adaptor molecule, myeloid differentiation protein (MyD88) and selected Toll-like receptors. Our goal is to assess the relative contribution of MyD88 and Toll-like receptors 2 and 4 in host response to H. pylori infection and to monitor T-cell responses. An in vitro approach was used to demonstrate that naive T cells could be induced to produce IL-17 when cocultured with DC exposed to H. pylori. The differentiation of naive T cells to Th-17 subset requires IL-6, TGF-beta and IL-23. We monitored supernatants of wt DC cultures exposed to H. pylori and use ELISA to detect the secretion of these differentiating cytokines. Increase in mRNA expression levels for Th-17 differentiation cytokines in DC from wt mice was detected via RT-PCR at selected time intervals post-infection. This increase in TH-17 differentiating cytokine expressions is MyD88 dependent. Animal studies confirmed an IL-17 local immune response and a significant increase in infiltration of CD4+ and CD8+ T-cells to H. pylori infection of gastric tissue. Flow cytometric studies of stomach infiltrates indicated that CD4+ T-cells were the primary T-cell subset involved in the IL-17 immune response. Immunohistochemistry analysis of stomach infiltrates in gastric tissues confirmed that CD4+ were the primary IL-17 producer in vivo. Real-time PCR analyses of mouse stomach tissues, three months post-infection, indicated an up-regulation of IL-17 and interferon gamma expression. IL-17 and interferon gamma expression was essentially abrogated in the stomach tissue of MyD88 and TLR2/TLR4 knockout mice. GM-CSF expression, stimulated by IL-17, was also increased in stomach tissues of mice infected with H. pylori. Our results clearly indicate that Helicobacter pylori induces IL-17 signaling in murine models. In collaboration with Dr. Griffin Rodgers research group, Molecular and Clinical Hematology Branch, NHLBI, we showed that olfactomedin 4 (OLFM4), a novel member of the olfactomedin-related glycoprotein superfamily, plays a role in the host immune response to H. pylori infection. OIFM4 is constitutively expressed in neutrophils and the gastrointestinal tract. We demonstrated that OLFM4 acts as anti-inflammatory agents after H. pylori infection. We generated OLFM4-/- mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. Histological examination of tissues including bone marrow, esophagus, small intestine and colon did not reveal any discernable abnormalities in OLFM4-/- mice. H. pylori colonization in the gastric mucosa of OLFM4-/- mice was significantly lower compared with wild-type littermates. Production and expression of proinflammatory cytokines/chemokines such as IL-1-beta;, IL-5, IL-12 p70, and MIP-1-alpha was increased in OLFM4-/- mice compared with infected controls. Dipeptidyl peptidase I (DPPI) was identified as a binding partner for OLFM4. Proteolytic activity of DPPI, cathepsin G and neutrophil elastase was significantly higher in neutrophils of OLFM4-/- infected mice compared with wild-type mice. Furthermore, OLFM4 appeared to negatively affect NF-kappaB activation during H. pylori infection via a direct interaction with nucleotide-binding oligomerization domain-1 (NOD1) and nucleotide-binding oligomerization domain-2 (NOD2) . These observations indicate that OLFM4 exerts considerable influence on the host defense against H. pylori infection acting through NOD1 and NOD2 mediated NF-Kappa B activation and subsequent cytokines and chemokines production, which in turn inhibit host immune response and contribute to gastric persistence of H. pylori